This invention relates to pulsed field gel electrophoresis of large DNA.
In the process of separating DNA molecules by electrophoresis, an electric field is applied across a gel to separate DNA molecules as they are moved by the field through the gel.
It is known to use the characteristics of the field established across the gel to control the electrophoresis for maximum separation. The fractionation of different molecular weight DNAs is presumably due to the sieving effect of the agarose gel matrix rather than differing electrophoresis mobilities of the DNAs as found in a free (completely liquid) medium.
In one prior art technique of electrophoresis that has been used for separating DNA, a static, unidirectional electric field is applied to a DNA sample resulting in the migration of the DNA molecules through the agarose. This technique is referred to as continuous field electrophoresis. Continuous field electrophoresis easily separates DNA molecules of size up to about 20,000 base pairs.
Continuous field electrophoresis has a disadvantage in that for DNA molecules of sizes above approximately 20,000 base pairs, separation becomes more difficult because the migration rate becomes independent of molecular size except at very low field voltages. At very low field voltages, separations take a long time. Usually, the practical upper limit is reached at a separation time of about two wee k s because DNA degrades at temperatures suitable for electrophoresis at times longer than this. This long time period is believed to be due to the extreme difficulty of totally eliminating minute amounts of nuclease from the system.
Several techniques are known to be successful in resolving larger chromosome fragments. Some of these techniques are successful in resolving chromosome fragments larger than 1 megabase in agarose gels . These techniques are different forms of pulsed field gel electrophoresis (PFGE) which is the resolution of large DNA molecules by periodically changing the electric field pattern during electrophoresis to produce DNA migration direction changes. These direction changes typically vary from close to 90 degrees to greater than 120 degrees. Sometimes these direction changes are curved loops, such as a sequence of curved segments with individual angles of arc. At other times, they are a zigzag path with concentrated angle changes of direction at the corners. The changes in field pattern reorient the DNA molecules and the separating medium, thus improving DNA separation.
In the prior art PFGE techniques, the pulse lengths relating to changing the field pattern are of sufficiently long duration to change the gross configuration of the DNA, being longer than one second in duration for the separation of large DNA. The changes in gross configuration are affected by the pulse duration and changes in direction and may vary from realigning direction of a substantially straight elongated strand to creating hooks or staircase-shaped strands.
Each of the prior art pulsed field techniques has the disadvantage of using a time duration for changing the field pattern that is in the order of a second or longer for separating large DNAs.
With pulsed field gel electrophoresis (PFGE) as usually practiced, it is easy to separate large DNA of size up to one million base pairs. Above this size it it becomes progressively more difficult. In order to get clear separations of larger DNA, the field voltage must be reduced and the angle-switching time increased. For example, the largest chromosome of the yeast Saccharomyces cerevisiae (YPH 80) about 1.5 megabase pairs, can be resolved in 15 hours with a field switching time of 120 seconds per direction and at a field voltage of 6 volts per centimeter. It is believed that this is an optimum separation. However, the S. pombe chromosomes of 3, 4.5 and 5.7 megabase pair require a switching time of 30 minutes per direction and a field of 115 volts per centimeter. The separating time is 3 to 6 days. It is believed that this is also an optimum separation. If the field voltage is raised in an attempt to get a faster separation, the DNA does indeed move faster but it smears out so that the bands overlap and no separation is discernible.
In the past, orthogonal pulses of duration too short to allow change in DNA configuration to take place were expected to appear as a vector sum, and be generally useless for separating DNA. This lack of separating effect was predicted in Schwartz, D.C. (1985) "Giga-Dalton Sized DNA Molecules ," p. 84, doctoral dissertation, Columbia University (University Microfilms International).
The prior art is deficient in some respects in providing adequate explanations of why pulsed field techniques provide the result that has been observed. As part of the development of the invention, a novel explanation has been developed.
According to this novel explanation, the limitations of ordinary pulsed field gel electrophoresis (PFGE) for separating very large DNA occurs because of the electric field and time dependent forking, tumbling and bunching motion of DNA in gel. These effects have been reported and recorded on videotape cited by Smith, S.B., Aldridge, P.K., and Calles, J.B. (1989); Science 243 203-206. The videotape shows actual motion of DNA in constant and varying electric fields: Smith, et al (1989); "Observation of Individual DNA Molecules Undergoing Gel Electrophoresis"; University of Washington Instructional Media Services, Source L66-79-90, University of Washington, Seattle, Wash. 98195.
DNA is negatively charged and therefore a straight length of DNA has a positive counterion sheath around it when in an aqueous buffer solution. The positive ion in buffer solutions used for DNA electrophoresis is Tris. Tris is selected as the positive ion because it does not bind to the DNA. A consequence of this is that Tris ions comprising the counterion sheath are free to move along the length of the DNA in response to electrical and thermal diffusion forces.
If the DNA molecule is located in an electric field parallel to its length, it starts to migrate straight toward the positive electrode because it has a negative charge. However, the counterion sheath has a positive charge and therefore tends to be repelled from the positive electrode. The counterion sheath does not leave the DNA because this would expose the negative charge of the DNA thus bringing the counter ion sheath back. However, the centroid of the counterion sheath shifts toward the trailing edge of the molecule.
Because the centroid of the counterion sheath shifts toward the trailing edge of the molecule, the trailing end of the molecule is surrounded with more positive charges than the leading end. The trailing end of the molecule is immersed within this concentrated charge and is surrounded by it. The leading edge of the molecule is outside of the concentrated charge area and "sees" the concentrated charge behind it. This decreases the net electric field on the leading edge of the molecule to a larger extent but decreases the net electric field on the trailing end of the molecule to a lesser extent.
Because the net electric field on the trailing edge is decreased less than the net electric field on the leading edge, the trailing end of the molecule tends to migrate faster than the leading end of the molecule, causing the bunching phenomenon reported by Smith and others. This effect may be aggravated in pulsed field gel electrophoresis when the DNA is long enough to be completely engaged in a previous corner-turning at the time the next corner turning starts. This is because the trailing, concentrated counterion sheath will be particularly concentrated at a previous sharp corner in the DNA where there are two continuous segments of it in close proximity.
When the DNA molecule bunches up and is no longer linear, its counterion polarizability decreases because of the decrease in effective length of the DNA. The DNA becomes more isotropic; more compact than elongated. The positive counterion sheath then becomes more isotropic, encouraging a leading end of the DNA to propagate out of the bunched up DNA, and eventually pulling out the bunched DNA into a more or less straight length of DNA.
As before, the counterion sheath becomes more concentrated at the trailing end of the length of DNA than the leading end, but because of the finite relaxation time of the counterion sheath it takes a while, such as 30 seconds, for it to do so. When the counterion sheath becomes sufficiently anisotropic, the DNA bunches up again and the cycle repeats. Under some circumstances, this phenomenon does not repeat at very regular, clock-like, intervals, and therefore, each DNA molecule can accumulate an error in its overall velocity compared to the average of the overall velocities of all the DNA of that species being separated.
This causes the observed band broadening and a smeared "nonseparation" if the effect is bad enough. Higher electric fields and longer free leading lengths of DNA cause more profound bunching problems. The foregoing can explain the faster separations possible at higher buffer concentrations than at lower buffer concentrations, because at higher buffer concentrations, counterions are known to form a thinner layer at the surface of a charged molecule. A thinner trailing counterion layer exerts less bunching influence at the leading end of the DNA.
Counterion sheath polarization also can explain snapback of the leading end of DNA undergoing electrophoretic migration as the field is removed. This snapback is shown in the Smith, et al. videotape. The effect may arise because the internal field from the displaced counterion sheath pulls back the leading end of the DNA before the counterion sheath anisotropy has time to relax upon external field removal. Smith attributes this effect to an "entropic spring" effect as the DNA pulls back into a random coil upon removal of the field. However, it is hard to see how this could cause rapid movement, since movement into a random coil is inhibited because the DNA is threaded through the pores of the gel.
The tendency toward bunching, combined with frictional forces, explains why it is more difficult to separate 6 megabase DNA than it is to separate 1 megabase DNA with ordinary pulsed field electrophoresis. In pulsed field gel electrophoresis, DNA is forced to turn successive sharp corners. The hydrodynamic or boundary friction drag retards the DNA molecule as it goes around each corner along its length, thus cumulatively tensioning the segment of the molecule behind the leading corner. This should be the case regardless of whether boundary friction or hydrodynamic friction is dominant at a corner.
Corner turning friction is believed to account for much of the differential mobilities of different lengths of DNA. The average number of corners turned per unit distance of migration is directly proportional to the length of the molecule. The frictional force increases as a rapid function of the sum of the number of corners turned. This is because, at the front-most or leading corner, frictional drag from the following corners along the length of the DNA adds additional tension which either increases boundary friction force at the leading corner or decreases the thickness of the hydrodynamic layer between the DNA and the gel strands defining the corner. A thinner hydrodynamic layer results in higher viscous friction forces between the gel strand and the DNA. The retardation force due to the corners turned by the DNA is an exponential function of the summation of the angles of all the corners. This follows from basic engineering theory relating to ropes on capstans, belts on pulleys, etc. Tensioning of the DNA around the leading corner arises from the pull of the electric field on the segment of DNA ahead of the leading corner, working with frictional drag on the segment of DNA behind the leading corner. If the turn angle at the leading corner exceeds 90 degrees, a component of the electric field will exert a reverse pull on the DNA segment behind the corner, adding to the tensioning at the corner. This can account for the fact that corner angles greater than 90 degrees (typically 120 degrees) work better than angles of 90 degress or less with pulsed field electrophoresis. It can be seen that alternate corners do not have this field tensioning effect at any one time. However, when the field makes one of its periodic direction changes, the corners which previously had no tensioning due to field effects become tensioned due to field effects. Conversely, corners which previously were tensioned at least partly due to field effects lose this component of tensioning. Since all corners have the same tensioning mechanisms, they can be considered as a group.
The reason that ordinary pulsed field electrophoresis can easily separate DNA of size greater than 20 kilobase pairs but less than one megabase pairs is that the leading end of a migrating DNA molecule is closer to the leading (most recently) turned corner than it is to the other end of its own length. Since the polarizability of the counterion sheath of a linear molecule is proportional to the cube of its length in the direction along the electric field, the effective polarizability of the free or leading length of the molecule is only the length from the leading end to the location of the most recent corner turning. With ordinary pulsed field electrophoresis, this should allow the use of higher electric fields and therefore faster migration rates compared to conventional constant field electrophoresis, with less serious problems with the bunching problem described previously.
The foregoing explanation is consistant with the increasing difficulty that is observed when trying to separate DNA molecules progressively larger than 1 megabase pairs in length with ordinary pulsed field electrophoresis.